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For the development of biosensors or microarrays in biotechnology functionalized surfaces e.g. aminated glass surfaces can be used. Knowledge of the quantity of primary reactive groups on such surfaces is indispensable for a defined secondary modification with commonly biochemical entities, eventually resulting in more reliably tailored surfaces with better controlled properties. Here, we investigated aminated glass surfaces with varying densities of amino groups prepared from binary mixtures of silanes. Subsequent labeling of the amino groups with a fluorophore containing a high number of fluorine atoms allows complementary quantification of the organic groups by optical fluorescence spectroscopy, X-ray Photoelectron Spectroscopy (XPS) and traceable Total-Reflection X-ray Fluorescence analysis (TXRF).
Reference-free TXRF with soft X-ray excitation determines the mass deposition of elements such as carbon, nitrogen and fluorine, yielding the areal density of primary functional groups on the surface. The TXRF-measurements were performed at the PTB beamline for undulator radiation at the electron storage ring BESSY II which provides monochromatic soft X-rays with high spectral purity and photon flux. This approach allows the calibration of optical fluorescence spectroscopy and XPS to deliver traceable quantitative data.